Each year, rotavirus gastroenteritis kills an estimated 600,000 children, worldwide, and hospitalizes approximately 60,000 children, in the U.S. Since the withdrawal of a rotavirus vaccine, it is again a major childhood illness for which no immunization is available. The process of cell entry by rotavirus is a major target for intervention against disease. To initiate infection, rotavirus translocates a large, transcriptionally active particle across a membrane and into the cytoplasm. The outer layer of the non-enveloped rotavirus particle is the delivery apparatus that accomplishes this poorly understood translocation. The outer capsid contains two proteins, VP7 and VP4, both of which participate actively in entry and are targets of neutralizing antibodies. VP7 causes uncoating on loss of calcium. VP4, a spike protein, is cleaved by trypsin to prime the virus for infection. VP4 is central to the mechanism of membrane penetration. It contains a head domain and a membrane interaction domain. We have purified uncleaved VP4, primed the purified protein with protease, determined the structure of the head domain, and crystallized the primed form of the membrane interaction domain. Using these crystals, we will solve the structure of the primed conformation of the membrane interaction domain. This structure will be used to refine strategies for the structural analysis of the uncleaved conformation of VP4. We will also determine the structures of functionally important VP4 variants. We have developed a "recoating genetics" system for rotavirus by recoating subviral particles with purified, recombinant VP4 and VP7, boosting infectivity by 4 to 5 orders of magnitude. This system will allow analysis of the effect of engineered mutations, including structure-based mutations, on the function of VP4 during cell entry. Mutational studies will clarify the determinants of sialic acid-dependence, protease priming, and membrane penetration. Cell culture-based assays using recoated particles will focus on separating carbohydrate, lipid, and protein binding during entry using structure-based mutations and on blocking entry-associated conformational changes by introducing reversible disulfide cross-links. These structural and functional studies will clarify the rotavirus entry pathway, provide a model for entry by non-enveloped viruses, and define the structural basis for rotavirus neutralization by antibodies against VP4.